Inhibition of SEMA3A in the prevention and treatment of ocular hyperpermeability

ABSTRACT

Described herein is a method of preventing or treating ocular vascular hyperpermeability including macular edema, in a subject comprising inhibiting Sema3A activity. Also disclosed are compositions and their use for preventing or treating Sema3A-dependent ocular vascular hyperpermeability.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Entry Application of PCT application noPCT/CA2014/050119 filed on Feb. 21, 2014 and published in English underPCT Article 21(2) which itself claims priority, under 35 U.S.C. §119(e),of U.S. provisional application Ser. No. 61/767,419, filed on Feb. 21,2013. All documents above are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

This application contains a Sequence Listing in computer readable formentitled “12810_553_ST25”, created on Aug. 4-18, 2015 having a size ofabout 40 Kbytes, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to ocular vascular hyperpermeability. Morespecifically, the present invention is concerned with the inhibition ofthe SEMA3A pathway for the prevention or treatment of macular edema.

BACKGROUND OF THE INVENTION

Diabetic retinopathy (DR) is the most prominent complication of diabetesand the leading cause of blindness in working age individuals^(1,2). Itis characterized by an initial microvascular degeneration followed by acompensatory but pathological hyper-vascularization mounted by thehypoxic retina in an attempt to reinstate metabolic equilibrium³⁻⁵.Although often initially asymptomatic, loss of sight is provokedprimarily by diabetic macular edema (DME), vitreal hemorrhages and inadvanced cases, pre-retinal neovascularization and tractional retinaldetachment^(6,7). Of these, DME is the main cause of central vision lossin diabetics⁸, affecting over 25% of patients suffering from diabetes.It is triggered secondary to the deterioration of the blood-retinalbarrier (BRB) and the consequent increase in extravasation of fluids andplasma components into the vitreous cavity. Ultimately, the decrease inretinal vascular barrier function leads to vasogenic edema andpathological thickening of the retina.

There are generally 3 stages to diabetic retinopathy: i)non-proliferative retinopathy (NPR); ii) macular edema; and iii)proliferative diabetic retinopathy.

The first stage of diabetic retinopathy, non-proliferative retinopathyor background retinopathy often has no noticeable signs or symptoms,although retinal swelling may be present. This is the stage where thetiny capillaries of the retina become semi-permeable membranes (Later,they will leak fluid and blood.). During the earliest stages, diabeticretinopathy is often asymptomatic. This means that there are nonoticeable symptoms—such as pain or vision loss- to the patient, but aneye specialist might find signs of the disease. For example, retinalswelling may be present, which can only be detected through an eyeexamination.

The second stage of diabetic retinopathy is macular edema. The macula isthe part of the retina responsible for sharp, direct vision due to itshigh density in cones photoreceptors. It is situated at the back of theretina. Macular edema refers to the accumulation of fluid within theretina at the macular area (distinct from the condition where the fluidaccumulates under the retina). The pathophysiology depends on theprimary cause but usually, the end-point is vascular instability and abreakdown of the blood-retinal barrier, leading to visual impairment.

When the center of the macula begins to swell, vision may become blurry.This middle stage of diabetic retinopathy may overlap the other stages.This is the stage where the blood-retinal barrier is compromised andcapillaries in the retina begin to leak fluid, causing swelling andblurred vision.

There are two types of macular edema: focal and diffuse. Focal macularedema occurs when the retinal capillaries develop micro-aneurisms whichleak fluid, resulting in several distinct points of leakage. Diffusemacular edema is caused by the dilation of retinal capillaries, creatingleakage that is diffused over a general area. The type of macular edemapresent will determine the kind of diabetic retinopathy treatment. Earlydetection of macular edema helps ensure the most effective treatment.

As the disease advances, minor visual impairment can occur. Althoughpatients are still able to see, they can be frustrated by blurring andblind spots that inhibit clear vision. These symptoms of diabeticretinopathy are sometimes linked to macular edema, which is the swellingof the part of the eye that controls central vision, known as themacula.

As damaged blood vessels begin to break, blood can leak into the eye.This third stage of diabetic retinopathy, called proliferative diabeticretinopathy (PDR), is characterized by cloudiness and impaired vision.When the retinal capillaries break, they are no longer able to supplythe retina with the necessary nutrients. The nutrient-starved retinasends out a chemical signal that prompts the growth of new capillaries.This growth is called neovascularization.

The new blood vessels that form as a result of proliferative diabeticretinopathy cause more damage to the eye. These capillaries are unableto restore nutrients to the retina because they are fragile and weak.They also tend to burst, causing blood and fluid to leak into the eye.The new vessels also exert traction on the surrounding structures andconnective tissue, which can eventually detach the retina. Intraocularpressure can also increase as a result of the new capillaries, as theycan block the ducts where fluid is drained from the eye. This conditionis known as neovascular glaucoma. During proliferative diabeticretinopathy, scar tissue development, retinal detachment, and blindnesscan occur.

If the disease has progressed into proliferative diabetic retinopathywithout the patient receiving any preventative care or medicalintervention, retinal detachment and blindness can result. At this time,PDR is the leading cause of new cases of blindness in the United States.Retinal detachment, macular edema, and the breakdown of capillaries inthe retina can all prevent normal blood flow through the eye and lead tototal vision loss.

Macular edema is not limited to the context of diabetes.Hyperpermeability of blood vessels and leakage of the blood-retinalbarrier can occur in a number of circumstances. The most frequent formof macular edema is cystoid macular edema, which is characterized byintraretinal edema contained in honeycomb-like spaces. CME is a commonpathological response to a variety of insults (e.g., followingintraocular (cataract) surgery, in central and branch retinal veinocclusions, following injury to the eye, in association with choroidaltumors or in various types of vascular retinal diseases or retinaldystrophies). CME is also one of the many conditions that may arise fromage-related macular degeneration.

Although significant effort has been invested in elucidating themechanisms that govern macular edema and in particular destructivepre-retinal neovascularization in DR^(6,9,10), considerably less isknown about the cellular processes that lead to increased retinalvascular permeability. Consequently, the current standards of carepresent non-negligible side-effects. These include increased cataractformation and a harmful rise in intraocular pressure with intravitrealuse of corticosteroid⁹. Similarly, anti-VEGF (vascular endothelialgrowth factor) therapies, which in general exhibit respectable safetyprofiles, may be associated with increased thromboembolic events¹¹,possible neuronal toxicity and geographic atrophy when used for longterm regiments^(12,13). Moreover, the first and most widely used form oftreatment is panretinal photocoagulation for either proliferativediabetic retinopathy (PDR) or grid/focal laser for DME. laser-basedphotocoagulation approaches destroy hypoxic retinal tissue secretingpro-angiogenic factors and inadvertently lead to reduced visual field orcentral or paracentral scotomas. These therapeutic limitations highlightthe need for novel pharmacological targets and interventions.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

Accordingly, Applicant has identified a novel therapeutic target,Sema3A, for the prevention and treatment of retinal vascularhyperpermeability related retinopathy including non-proliferativediabetic retinopathy and macular edema.

Sema3A is a classical neuronal guidance cue also involved in a varietyof cellular responses through its binding to Neuropilin-1 (Nrp-1), anon-tyrosine kinase multifunctional receptor. Neuropilin-1 has theparticular ability to bind two structurally dissimilar ligands viadistinct sites on its extracellular domain¹⁵⁻¹⁷. It binds Sema3A^(18,19)provoking cytoskeletal collapse and VEGF₁₆₅ ^(16,17,19,20) enhancingbinding to VEGFR2 and thus increasing its angiogenic potential²¹.Crystallographic evidence revealed that VEGF₁₆₅ and Sema3A do notdirectly compete for Nrp-1 but rather can simultaneously bind to Nrp-1at distinct, non-overlapping sites²². Moreover, genetic studies showthat Nrp-1 distinctly regulates the effects of VEGF and Sema3A onneuronal and vascular development²³. Notably, it was proposed that,similar to VEGF, Sema3A may itself promote vascular permeability(Acevedo et al., 2008); this is a counterintuitive observation, giventhe divergent biological roles of VEGF and Sema3A. However, the role ofSema3A in mediating the breakdown of barrier function, such as thatobserved in diabetic retinopathy, has not been explored to date.

Applicant show herein for the first time that Sema3A is involved in thedeterioration of the blood-retinal barrier (BRB) function in diabeticretinopathy. Applicant demonstrates in both human patients and animalmodels that ocular Sema3A is robustly induced in the early stages ofdiabetes (prior to VEGF inducement). Applicant further shows that SEMA3Amediates, via NRP1, the breakdown of the inner BRB, leading to increasedvascular permeability thereby contributing to retinal swelling andmacular edema. Accordingly SEMA3A provides a good target for theprevention of symptoms associated with macular edema or for earlytreatment of the disease (e.g., in the non-proliferative stage ofdiabetic retinopathy), prior to substantial pathologicalneovascularization and damages to the retina. Neutralizing Sema3A thusrepresents an attractive alternative therapeutic strategy to counterpathologic vascular permeability in DR.

Accordingly, in a first aspect, the present invention provides a methodof preventing or treating macular edema in a subject comprisinginhibiting Sema3A-mediated cellular activity.

In a related aspect the present invention provides a method ofpreventing or treating non-proliferative diabetic retinopathy in asubject comprising inhibiting Sema3A-mediated cellular activity.

In another aspect, the present invention provides a method of preventingor treating retinal swelling in a subject comprising inhibitingSema3A-mediated cellular activity.

In an embodiment, the Sema3A-mediated cellular activity comprisesSema3A-mediated vascular permeability. In a related embodiment, theSema3A-mediated activity comprises Sema3A binding to the Nrp-1 receptor.

In an embodiment the macular edema is substantially non-proliferativemacular edema. In another embodiment, the macular edema is diabeticmacular edema. In another embodiment the diabetic macular edema issubstantially non-proliferative (i.e., neovascularization issubstantially low or absent). In yet a further embodiment, the macularedema is age-related macular edema. In an embodiment, the age relatedmacular edema is substantially non-proliferative.

In an embodiment, the methods of the present invention compriseadministering a therapeutically or prophylactically effective amount ofa Sema3A antagonist to the subject. In an embodiment, the antagonistreduces Sema3A nucleic acid or protein expression. In anotherembodiment, the Sema3A antagonist reduces Sema3A secretion. In a furtherembodiment the Sema3A antagonist reduces Sema3A vitreal concentration.In a further embodiment the Sema3A antagonist reduces Npr-1 ocular(e.g., vitreal) concentration and/or activity. In yet anotherembodiment, the Sema3A antagonist inhibits Sema3A-mediated cellsignaling. In an embodiment, the Sema3A-mediated cell signalingcomprises binding of Sema3A to its cognate receptor Npr-1.

In an embodiment, the antagonist is an anti-Sema3A antibody. In anembodiment, the anti-Sema3A antibody specifically inhibits Sema3Abinding to Nrp-1 but does not substantially reduce VEGF binding toNrp-1. In another embodiment, the Sema3A antagonist is an Nrp-1 antibodythat inhibits binding of Sema3A to the receptor. In a preferredembodiment, the Nrp-1 antibody does not substantially reduce VEGFbinding to Nrp-1. In a particular embodiment, the Nrp-1 antibody bindsto the a1, a2 or a1/a2 domain of Nrp-1.

In yet a further embodiment, the Sema3A antagonist is a soluble Nrp-1polypeptide or fragment thereof that binds to Sema3A. In an embodiment,the fragment comprises domain a1, a2 or a1 and a2 of Nrp-1. In anembodiment, the fragment does not comprise domains b1, b2 or IA and b2of Npr-1. In a related embodiment, the soluble Nrp-1 fragment does notsubstantially bind to VEGF. In an embodiment, the fragment comprisesdomains a1a2 and b1b2 or portions thereof and binds to Sema3A and VEGF.

In another embodiment, the Sema3A antagonist reduces Sema3A or Npr-1nucleic acid or protein expression. In an embodiment, the Sema3Aantagonist is a Sema3A shRNA or antisense. In an embodiment, the Sema3Aantagonist is a Npr-1 shRNA or antisense that binds to a polynucleotideencoding a Npr-1 polypeptide, preferably a human Npr-1 polypeptide(e.g., SEQ ID NO:2 or 12).

In a further aspect, the present invention concerns a composition forreducing retinal vascular hyperpermeability comprising one or more ofthe above-described Sema3A antagonist together with a suitablepharmaceutical carrier.

In yet another aspect, the present invention concerns a composition forthe prevention or treatment of vascular hyperpermeability, diabeticretinopathy, macular edema, preferably, age related macular edema, morepreferably non-proliferative age-related macular edema and even morepreferably, non-proliferative diabetic macular edema comprising one ormore of the above-described Sema3A antagonist together with a suitablepharmaceutical carrier.

In a preferred embodiment, the compositions of the present invention aresuitable for intraocular administration. In an embodiment, thecompositions are formulated in the form of eye drops. In anotherembodiment, the compositions are formulated for intraocular injection.

In an embodiment, the composition comprises one or more additionalactive agent useful in the treatment of non-proliferative diabeticretinopathy or macular edema.

In a related aspect, the present invention also concerns the use of atherapeutically or prophylactically effective amount of one or more ofSema3A antagonists of the present invention for reducing retinalvascular hyperpermeability in a subject. In an embodiment, the use isfor the prevention or treatment of non-proliferative diabeticretinopathy. In another embodiment, the use is for the prevention ortreatment of macular edema. In an embodiment, the macular edema isdiabetic macular edema. In an embodiment, the diabetic macular edema issubstantially free of neovascularization (i.e., it is mainlynon-proliferative). In a further embodiment, the edema is age-relatedmacular degeneration. In another embodiment, the age-related macularedema is substantially free of neovascularization.

In an embodiment, the above mentioned subject suffers from early stagesof diabetes. In an embodiment, the subject suffers from type 1 diabetesmellitus (T1DM). In another embodiment, the subject suffers from type 2diabetes mellitus. In an embodiment, the subject's vision is normal(he/she is asymptomatic i.e., does not suffer from symptoms associatedwith macular edema of vascular hyperpermeability such as spotted orblurry vision). In another embodiment, the subject does not suffer fromsubstantial pericytes loss. In an embodiment, the subject has beendiagnosed with non-proliferative diabetic retinopathy or macular edema.In an embodiment, the subject suffers from retinal swelling or retinalvascular hyperpermeability. In a specific embodiment, the subject issuffering from blood retinal barrier swelling.

In an embodiment, the Sema3A antagonist is administered prior to theonset of substantial macular edema. In another embodiment, the Sema3Aantagonist is administered prior to the onset of blurry or spottedvision. In another embodiment, the Sema3A antagonist is administeredprior to VEGF inducement (i.e., prior to an increase in VEGFexpression). In another embodiment, the Sema3A antagonist of the presentinvention is administered in combination with one or more other drugsused for the prevention and/or treatment of macular edema and/ordiabetes. Non-limiting examples of drugs used for the treatment ofmacular edema comprises bevacizumab (Avastin™), Ranibuzimad (Lucentis™),aflibercept (Eylea™) and corticosteroids. The present invention alsoconcern compositions comprising a Sema3a antagonist alone or incombination with one or more drugs used for the treatment of macularedema and diabetic retinopathy.

Having demonstrated that increased Sema3A activity is associated withthe BRB leakage and retinal vascular hyperpermeability, the inventionrelates to the use of Sema3A as a target in screening assays used toidentify compounds that are useful for the prevention or treatment ofretinal vascular hyperpermeability (e.g., non-proliferative diabeticretinopathy and macular edema), said method comprising determiningwhether:

-   -   (a) the level of expression of a Sema3A nucleic acid or encoded        polypeptide;    -   (b) the level of Sema3A activity;    -   (c) the level of a molecule generated by a Sema3A activity; or    -   (d) any combination of (a) to (c);

is decreased in the presence of a test compound relative to in theabsence of the test compound; wherein the decrease is indicative thatthe test compound is potentially useful for the prevention and treatmentof retinal vascular hyperpermeability. In an embodiment, theabove-mentioned method is an in vitro method. In an embodiment, theSema3A activity is its binding to the Nrp-1 receptor. In a furtherembodiment, the Sema3A activity is the increased vascular permeability.

The present invention also relates to a method of identifying orcharacterizing a compound for preventing or treating retinal vascularhyperpermeability comprising:

-   -   a) contacting a test compound with a cell comprising a first        nucleic acid comprising a transcriptionally regulatory element        (e.g., endogenous promoter or fragment thereof) normally        associated with a Sema3A gene, operably-linked to a second        nucleic acid comprising a reporter gene capable of encoding a        reporter protein; and    -   b) determining whether the reporter gene expression or reporter        activity is decreased in the presence of the test compound;        wherein a decrease in the reporter gene expression or reporter        gene activity is indicative that the test compound may be used        for decreasing vascular hyperpermeability (e.g., treating or        preventing non-proliferative diabetic retinopathy and macular        edema).

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 shows elevated Sema3A levels in the vitreous of human T1 DMpatients suffering from diabetic retinopathy. (a) Western blot (Wb)analysis revealed that both pro-(˜125 kDa) and active (˜95 kDa) forms ofSema3A were robustly induced in patients affected by Type 1 DiabetesMellitus. (b) Wb quantification, ˜125 kDa Sema3A signal was ˜250-foldhigher in DR relative to controls (p<0.05); ˜95 kDa Sema3A signal was˜175-fold in DR patients (p<0.05). (c,d) Optical Coherence Tomography(OCT) revealed significant retinal swelling, mostly in the macular andperi-macular zones. (e) Detailed patient characteristics;

FIG. 2 shows that neuronal Sema3A is upregulated in the early phases ofSTZ-induced diabetes and its expression is geographically consistentwith a role in macular edema. (a) Streptozotocin (STZ) was administeredto ˜6 week-old C57BL/6J mice and glycemia monitored according to thescheme; a mouse with non-fasted glycemia higher than 17 mM (300 mg/dL)was considered diabetic. (b) At 4 weeks after induction of diabetes,retinal Sema3A mRNA levels rose more than 2-fold in STZ treated micewhen compared to vehicle-injected controls (P=0.0045, n=5), ˜3-fold at 8weeks (P=0.0011, n=8), 4-fold at 12 weeks (P=0.00846, n=4) and ˜2.5 foldat 14 weeks (P=0.0334, n=3). Conversely, VEGF levels remained unchangeduntil 14 weeks where they rose by ˜3-fold (P=0.0253, n=3). (c)Pericyte-specific staining of smooth muscle actin (SMA) in STZ- andvehicle-treated mice showing that Sema3A expression preceded pericyteloss. (d) Pathologically elevated blood glucose ˜30 mM (p<0.0001, forboth time points) at both 4 and 8 weeks of diabetes, STZ-treated mice.(e) At 8 weeks, tight junction (TJ) component occludin mRNA levelsremained unchanged, whereas claudin-5 decreased by 38.6% (p<0.01). (f)Immunohistochemistry of Sema3A on retinal cryosections andco-localization with the retinal ganglion cell (RGC) markerpIII-tubulin, of the ganglion cell layer (GCL) and inner-nuclear layer(INL). (g) Laser-capture micro-dissection of retinal layers from normaland diabetic mice. (h) Quantitative RT-PCR for Sema3A on retinal layersof normal and diabetic mice.

FIG. 3 shows that the retinal barrier function is compromised by Sema3A.(a) Intravitreal injection of Sema3A resulted in a ˜2-fold increase(p<0.01) in retinal vascular permeability (VP) as determined by EvansBlue (EB) permeation; a similar increase was observed with intravitrealadministration of VEGF (p<0.05) and with a combination of both Sema3Aand VEGF (p<0.01). (b) Confocal images of retinal sections injected withvehicle, VEGF and SEMA3A, showing the representative pattern ofincreased EB leakage. (c) Trans-endothelial resistance measured by ECISdemonstrates that Sema3A effectively reduces endothelial barrierfunction. (d) Western blot (WB) analysis of Human Retinal MicrovascularEndothelial Cells (HRMECs); treatment with either Sema3A or VEGF lead torobust phosphorylation of Src at Tyr416; FAK was phosphorylated onTyr576 and 577 (sites for Src-kinases); the adherence junction proteinVE-cadherin became phosphorylated respectively on tyrosine-731 (pY731),site associated with increased VP; an additive or enhanced effect wasnot observed when simulation was performed with a combination of Sema3Aand VEGF, suggesting action via redundant pathways. (e) Schematicrepresentation of Sema3A signaling leading to VE-cadherinphosphorylation and tight junctions loosening. (f) Confocal microscopyof Sema3A-treated HRMECs revealed formation of vascular retractionfibers as determined by VE-cadherin and phalloidin staining (whitearrows; FIG. 3f ); retraction was similar to that with VEGF alone orwith a combination of VEGF and Sema3A. (g) Flatmounted retinas injectedwith Sema3A or VEGF showed higher VE-cadherin phosphorylation at Y731(white arrows) than vehicle-injected retinas in colocalization withretinal vessels—lectin stain—. (h) Retinal flatmounts from STZ-injectedand vehicle-injected mice. (i) Cell death and apoptosis by caspase 3assessment following Sema3A treatment (100-200 μM).

FIG. 4 shows that targeted silencing of neuron-derived Sema3A andintravitreal neutralization of Sema3A efficiently reduce vascularpermeability in T1 DM. (a) Lentiviral vectors with a VSVG capsid exhibithigh tropism for RGCs and cells of the ONL when deliveredintravitreally, as depicted by Lv vector carrying GFP RNA.; Lv.shRNAagainst Sema3A was used to specifically block Sema3A production in RGCsor neurons of the INL in vivo. (b) While STZ-treated mice show a 56.8%increase in permeability (assessed by Evans Blue permeation, (p<0.05)),(c) a single intravitreal injection of Lv.shSema3A at 5 weeks ofdiabetes lead to a significant 62.3% reduction in retinal Sema3Aexpression (p<0.005) and (d) provoked a proportional 49.5% decrease invascular leakage (p<0.05). (e) To neutralize vitreal Sema3A, we usedrecombinant (r) soluble Nrp-1 as a bivalent trap for both Sema3A andVEGF. Neuropilin-1 is a single-pass receptor with its extracellulardomain subdivided into distinct sub-domains of which a1a2 bindsemaphorin and b1b2 bind VEGF. (f) Intravitreal injection of rmNRP1 inSTZ mice at weeks 6 and 7 after induction of diabetes lead to a 48.1%reduction in retinal permeability at week 8 of diabetes (P=0.012, n=6(18 mice)). Conversely, injection of a neutralizing antibody againstmouse VEGF was ineffective at reducing diabetes-induced retinalpermeability at this stage of disease when compared to vehicle(P=0.7302, n=5 (14 mice)). Values expressed relative to vehicle injectedretinas;

FIG. 5 shows that conditional knockout of Nrp-1 prevents Sema3A-inducedretinal barrier function breakdown. Systemic administration of tamoxifenduring a 5 day period effectively deleted Nrp-1 protein (a) and gene (b)expression. (c) In absence of NRP1, intravitreally administered Sema3Adid not increase vascular leakage (P=0.36; n=7 (21 mice)), whileTam-treated TgCre-ESR1/Nrp1+/+ controls show 3-fold higher vascularleakage (P=0.00065; n=3 (9 mice)). (d) Conversely, disruption of Nrp1did not influence VEGF-induced vascular retinal permeability (p=0.0024;n=3 (9 mice)), suggesting that VEGF-induced retinal vascular leakage isindependent of NRP1 as previously reported.;

FIG. 6 shows human Sema3A precursor protein sequence (SEQ ID NO:1). Thissequence is further processed into mature form. Residues 1-20 correspondto the signal peptide;

FIG. 7 shows human soluble Neuropilin-1 (Nrp-1) receptor proteinsequence (GenBank Acc. No. AAH07737.1-SEQ ID NO:2) and

FIG. 8 shows an alignment between rat (SEQ ID NO:15, Access. Nos.EDL96784, NP 659566), human (SEQ ID NO: 12, Accession No. NP_003864) andmouse (SEQ ID NO: 14, Accession No. ACCESSION NP_032763) Nrp-1 togetherwith signal domain, Sema3a binding domains a1a2, VEGF binding domainsb1b2, domain C, cytoplasmic domain and transmembrane domain.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The deterioration of the blood retinal barrier and consequent macularedema is a cardinal manifestation of diabetic retinopathy (DR) and theclinical feature most closely associated with loss of sight. Whilemacular edema affects over 25% of patients suffering from diabetes,currently available treatment modalities such as locally administeredcorticosteroids and recently approved anti-VEGF therapies, presentseveral drawbacks. Here Applicant provides the first evidence from bothhuman and animal studies for the role of the classical neuronal guidancecue Semaphorin3A in instigating pathological macular vascularpermeability in type I diabetes. While classically associated withembryogenesis and neuronal and vascular patterning, investigation of thedynamics of expression reveal that Semaphorin3A is also induced in theearly hyperglycemic phases of diabetes within the neuronal retina andprecipitates initial breakdown of endothelial barrier function. Usingthe streptozotocin mouse model as a proxy for human diabeticretinopathy, Applicant demonstrates by a series of orthologousapproaches (gene silencing or treatment with soluble Neuropilin-1employed as a Semaphorin3A trap), that neutralization of Semaphorin3Aefficiently prevents retinal vascular leakage. The increase inpermeability provoked by Semaphorin3A is mediated through its cognatereceptor, Neuropilin-1. Conditional knockout of Neuropilin-1 inTg^(Cre-Esr1)Nrp1^(flox/flox) mice diminishes Semaphorin3A-inducedocular permeability. The present findings identify a new therapeutictarget for the prevention or treatment of non-proliferative retinopathy,macular edema and in particular DME.

Definitions

As used herein, the term Sema3A refers to Sema3A (e.g., HGNC: 10723;Entrez Gene: 10371; Ensembl: ENSG00000075213; OMIM: 603961; UniProtKB:Q14563;-FIG. 6, SEQ ID NO:1) and its functional isoforms, andallelic/polymorphic variants. Sema3A encodes a protein with an Ig-likeC2-type (immunoglobulin-like) domain, a PSI domain and a Sema domain.This secreted protein can function as either a chemorepulsive agent,inhibiting axonal outgrowth and neovascularization, or as achemoattractive agent, stimulating the growth of apical dendrites. It isexpressed in various tissues including stressed retinal ganglionneurons.

“Sema3A-mediated cellular activity” refers in general to thephysiological or pathological events in which Sema3A has a substantialrole. Non-limiting examples of such activities include i) deteriorationof the blood retinal barrier; ii) increased vascular permeability (i.e.,hyperpermeability); iii) inhibition of VEGF-induced neovascularizationat a hypoxic site (anti angiogenic effect); and modulation of axonalgrowth (e.g., inducement of growth cone collapse). Sema3A binds to theNeuropilin-1 receptor (Nrp-1).

As used herein, the term “Neuropilin-1 receptor” or “Nrp-1” receptorrefers to neuropilin-1 and its isoforms, and allelic/polymorphicvariants involved in Sema3A binding and signal transduction (e.g., HGNC:8004; Entrez Gene: 8829; Ensembl: ENSG00000099250; OMIM: 602069; andUniProtKB: 014786; FIG. 7, SEQ ID NO:2, SEQ ID NO:12). The basicstructure of neuropilin-1 comprises 5 domains: three extracellulardomains (a1a2, b1b2 and c), a transmembrane domain and a cytoplasmicdomain (See FIG. 8 and SEQ ID NO:12). The a1a2 (SEQ ID NO:13) domain ishomologous to complement sedxw234weqqcomponents C1r and C1s (CUB) whichgenerally contain 4 cysteine residues forming disulfide bridges. Thisdomain binds Sema3A. There exists several splice variants isoforms andsoluble forms of neuropilin-1 which are all encompassed by the presentinvention.

As used herein, “functional fragment” or “functional variant” (e.g., afunctional fragment of soluble Nrp-1 polypeptide or polynucleotide ofthe present invention) refers to a molecule which retains the sameactivity as the original molecule but which differs by anymodifications, and/or amino acid/nucleotide substitutions, deletions oradditions (e.g., fusion with another polypeptide). Modifications canoccur anywhere including the polypeptide/polynucleotide backbone (e.g.,the amino acid sequence, the amino acid side chains and the amino orcarboxy termini). Such substitutions, deletions or additions may involveone or more amino acids or in the case of polynucleotide, one or morenucleotide. The substitutions are preferably conservative, i.e., anamino acid is replaced by another amino acid having similarphysico-chemical properties (size, hydrophobicity, charge/polarity,etc.) as well known by those of ordinary skill in the art. Functionalfragments of the soluble Nrp-1 (SEQ ID NO:2) receptor include a fragmentor a portion of a soluble Nrp-1 polypeptide (e.g., the a1a2 domain, SEQID NO:13) or a fragment or a portion of a homologue or allelic variantof a Nrp-1 which retains inhibiting activity, i.e., binds to Sema3A andinhibits the transduction of Sema3A-mediated cellular activity. In aparticular embodiment, the Sema-3A-mediated cellular activity isvascular hyperpermeability. In an embodiment, the Npr-1 polypeptide isat least 80, 85, 88, 90, 95, 98 or 99% identical to SEQ ID NO:2. In anembodiment, the Npr-1 polypeptide is at least 80, 85, 88, 90, 95, 98 or99% identical to domains a1, a2, IA and/or b2 of Npr-1 as depicted inFIG. 8. In an embodiment, the Npr-1 is a functional variant whichincludes variations in amino acids which are not conserved between rat,mouse and human Nrp-1 as depicted in FIG. 8. Preferably, the Npr-1polypeptide/polynucleotide or fragment thereof is human.

In further embodiments, polypeptides and nucleic acids which aresubstantially identical to those noted herein may be utilized in thecontext of the present invention.

“Homology” and “homologous” refers to sequence similarity between twopeptides or two nucleic acid molecules. Homology can be determined bycomparing each position in the aligned sequences. A degree of homologybetween nucleic acid or between amino acid sequences is a function ofthe number of identical or matching nucleotides or amino acids atpositions shared by the sequences. As the term is used herein, a nucleicacid/polynucleotide sequence is “homologous” to another sequence if thetwo sequences are substantially identical and the functional activity ofthe sequences is conserved (as used herein, the term ‘homologous’ doesnot infer evolutionary relatedness). Two nucleic acid sequences areconsidered substantially identical if, when optimally aligned (with gapspermitted), they share at least about 50% sequence similarity oridentity, or if the sequences share defined functional motifs. Inalternative embodiments, sequence similarity in optimally alignedsubstantially identical sequences may be at least 60%, 70%, 75%, 80%,85%, 90%, 95%, 98% or 99% identical. As used herein, a given percentageof homology between sequences denotes the degree of sequence identity inoptimally aligned sequences. An “unrelated” or “non-homologous” sequenceshares less than 40% identity, though preferably less than about 25%identity, with any of SEQ ID NOs 1-14.

Substantially complementary nucleic acids are nucleic acids in which thecomplement of one molecule is substantially identical to the othermolecule. Two nucleic acid or protein sequences are consideredsubstantially identical if, when optimally aligned, they share at leastabout 70% sequence identity. In alternative embodiments, sequenceidentity may for example be at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99%. Optimalalignment of sequences for comparisons of identity may be conductedusing a variety of algorithms, such as the local homology algorithm ofSmith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, thesearch for similarity method of Pearson and Lipman, 1988, Proc. Natl.Acad. Sci. USA 85: 2444, and the computerised implementations of thesealgorithms (such as GAP, BESTFIT, FASTA and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, Madison, Wis.,U.S.A.). Sequence identity may also be determined using the BLASTalgorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10(using the published default settings). Software for performing BLASTanalysis may be available through the National Center for BiotechnologyInformation (through the internet at www.ncbi.nlm.nih.gov/). The BLASTalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence that eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighbourhood word score threshold. Initial neighbourhood wordhits act as seeds for initiating searches to find longer HSPs. The wordhits are extended in both directions along each sequence for as far asthe cumulative alignment score can be increased. Extension of the wordhits in each direction is halted when the following parameters are met:the cumulative alignment score falls off by the quantity X from itsmaximum achieved value; the cumulative score goes to zero or below, dueto the accumulation of one or more negative-scoring residue alignments;or the end of either sequence is reached. The BLAST algorithm parametersW, T and X determine the sensitivity and speed of the alignment. TheBLAST program may use as defaults a word length (W) of 11, the BLOSUM62scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of bothstrands. One measure of the statistical similarity between two sequencesusing the BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. In alternativeembodiments of the invention, nucleotide or amino acid sequences areconsidered substantially identical if the smallest sum probability in acomparison of the test sequences is less than about 1, preferably lessthan about 0.1, more preferably less than about 0.01, and mostpreferably less than about 0.001.

An alternative indication that two nucleic acid sequences aresubstantially complementary is that the two sequences hybridize to eachother under moderately stringent, or preferably stringent, conditions.Hybridisation to filter-bound sequences under moderately stringentconditions may, for example, be performed in 0.5 M NaHPO4, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1%SDS at 42° C. (see Ausubel, et al. (eds), 1989, Current Protocols inMolecular Biology, Vol. 1, Green Publishing Associates, Inc., and JohnWiley & Sons, Inc., New York, at p. 2.10.3). Alternatively,hybridization to filter-bound sequences under stringent conditions may,for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C.,and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel, et al. (eds),1989, supra). Hybridization conditions may be modified in accordancewith known methods depending on the sequence of interest (see Tijssen,1993, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York). Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point forthe specific sequence at a defined ionic strength and pH. For example,in an embodiment, the Sema3A antagonist is an antisense/RNAi or shRNAthat hybridizes to an Npr-1 nucleic acid sequence (preferably a humansequence).

As used herein the term “treating” or “treatment” in reference tomacular edema and/or non-proliferative retinopathy is meant to refer toa reduction/improvement in one or more symptom of macular edema and/ornon-proliferative diabetic retinopathy including but not limited tovision impairment (e.g., blind spots, spotty or blurry vision), retinalswelling, macular edema, vascular hyperpermeability; blood retinalbarrier integrity, retinal thickening, pericytes loss and/or presence ofcircinate rings of hard exudates.

As used herein the term “preventing” or “prevention” in reference tomacular edema or vascular hypermeability is meant to refer to areduction in the progression or a delayed onset of at least one of avision impairment (e.g., blind spots, spotty or blurry vision), retinalswelling, macular edema, vascular swelling/leakage, blood retinalbarrier integrity, retinal thickening, pericytes loss and/or presence ofcircinate rings of hard exudates.

As used herein, the term vascular hyperpermeability refers to anabnormal increase of the permeability of blood vessels and/orcapillaries compared to normal conditions e.g., in non-diabetic patientsor patients not suffering from any form of macular edema or retinalswelling. Vascular hyperpermeability may be acute (transient) orchronic. As a result of vascular hyperpermeability fluid moves from theblood stream past the blood vessels walls, thereby forming an area ofedema. In the context of the present invention, vascularhyperpermeability include swelling (e.g., retinal swelling) and abnormalleakage of the blood vessels including through the blood retinalbarrier.

As used herein the term “Sema3A inhibitor” or “Sema3A antagonist” refersto an agent able to reduce or block Sema3A-mediated cell signaling.Non-limiting examples include an agent which reduces or blocks theexpression (transcription or translation) of Sema3A, an agent able toreduce or block Sema3A secretion or an agent able to reduce or blockSema3A binding to its receptor Nrp-1 and an agent which reduce or block(transcription or translation) of Npr-1. Without being so limited, theagent can be natural or synthetic and can be a protein/polypeptide suchas but not limited to an antibody that specifically binds to Sema3A orNrp-1 receptor; a soluble Nrp-1 polypeptide or fragment thereof, apeptide, a small molecule, a nucleotide such as but not limited to anantisense or a shRNA specific to Sema3A nucleic acid sequence encoding aSema3A protein (e.g., SEQ ID NO:1) or Npr-1 nucleic acid sequence (GeneID 8829 (human), Gene ID 18186 (mus musculus) or GeneID 246331 (rattusNorvegicus) encoding a Npr-1 protein (e.g., SEQ ID NO:2 or 12). In anembodiment, the agent is able to prevent Sema3A-mediated cell signalingwithout substantially reducing VEGF binding to the Nrp-1 receptor andthus VEGF-mediated cellular signaling.

Methods, compositions, uses and packages of the present invention areparticularly useful for mammals and preferably humans. In a particularembodiment, the subject to which the Sema3A inhibitor of the presentinvention is administered suffers from diabetes. In another embodiment,the subject is at risk of suffering from diabetes. In an embodiment, thediabetes is Type 1 diabetes mellitus (T1DM). In an embodiment, thesubject has been diagnosed with macular edema or is at risk of sufferingfrom macular edema. In an embodiment, the macular edema is diabeticmacular edema. In an embodiment the macular edema is diffuse macularedema. In another embodiment, the macular edema is focal macular edema.In an embodiment, the subject suffers from non-proliferative retinopathy(i.e., pathological neovascularization is absent or substantially low atthe time the Sema3A inhibitor is administered). In an embodiment, thesubject is suffering from early stage diabetes. In a related embodiment,the subject has an increased blood glucose level compared to a healthysubject. In yet another embodiment, the subject does not suffer from asubstantial loss of pericytes. In yet another embodiment, the diabeticsubject suffers from retinal swelling.

As used herein, the expression “early stage diabetes” or the like meansthat the subject is still at an early stage of diabetes e.g., stages1-4, preferably, 1-3. Stage 1 is characterized by compensation: insulinsecretion increases to maintain normoglycemia in the face of insulinresistance and/or decreasing β-cell mass. This stage is characterized bymaintenance of differentiated function with intact acuteglucose-stimulated insulin secretion (GSIS). Stage 2 occurs when glucoselevels start to rise, reaching! 5.0-6.5 mmol/l; this is a stable stateof β-cell adaptation with loss of β-cell mass and disruption of functionas evidenced by diminished GSIS and ι β-cell dedifferentiation. Stage 3is a transient unstable period of early decompensation in which glucoselevels rise relatively rapidly to the frank diabetes of stage 4, whichis characterized as stable decompensation with more severe β-celldedifferentiation. Finally, stage 5 is characterized by severedecompensation representing a profound reduction in β-cell mass withprogression to ketosis. Movement across stages 1-4 can be in eitherdirection. For example, individuals with treated type 2 diabetes canmove from stage 4 to stage 1 or stage 2. For type 1 diabetes, asremission develops, progression from stage 4 to stage 2 is typicallyfound (see Diabetes 53 (Suppl. 3):S16-S210, 2004, which is incorporatedherein by reference in its entirety)

As used herein “pharmaceutically acceptable carrier” or “excipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, physiological media, and the like that arephysiologically compatible. In embodiments the carrier is suitable forocular administration. Pharmaceutically acceptable carriers includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents, such as for ocularapplication, is well known in the art. Except insofar as anyconventional media or agent is incompatible with the compounds of theinvention, use thereof in the compositions of the invention iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

Methods of Treating or Preventing Vascular Hyperpermeability

In a first aspect, the present invention concerns a therapeutic approachto the inhibition of vascular hyperpermeability and the formation ofmacular edema in subjects by administering a compound that specificallyinhibit Sema3A-mediated cellular activity. Sema3A-mediated cellularactivity can be inhibited by a number of approaches. Inhibition ofSema3A cellular activity may be done directly by reducing Sema3A nucleicacid or protein expression or by inhibiting the binding of Sema3A to itsassociated receptor, Nrp-1. Inhibition of Sema3A activity may also beachieved indirectly by targeting one of Sema3A known downstreameffectors (e.g., by targeting the Nrp-1 receptor) involved inSema3A-induced vascular hyperpermeability. Non-limiting examples ofapproaches for inhibiting Sema3A-mediated cellular activity include i)antibodies specific for Sema3A; ii) antibodies specific for Nrp-1 (i.e.,competing with Sema3A binding to the receptor); ii) by antisense andRNAi methods for reducing Sema3A expression and iv) by providing asoluble Nrp-1 receptor or fragment thereof, acting as a functionalSema3A trap.

Inhibition of Sema3A-Mediated Cellular Activity

a. Antibodies

In a particular aspect of the present invention, Sema3A cellularactivity (e.g., Sema3A-mediated-vascular hyperpermeability) can beinhibited by using Sema3A antibodies. In a particular embodiment, theseantibodies bind to the portion of Sema3A which interacts with itscognate receptor, Nrp-1, thereby preventing Sema3A-mediated cellularsignaling⁴¹.

Alternatively, antibodies directly targeting the Nrp-1 receptor, whichblock the binding of Sema3A binding to Nrp-1 may also be used. In aparticular aspect of the present invention, antibodies targeting Nrp-1block Sema3A binding to the receptor but do not substantially interferewith VEGF binding to Nrp-1. In an embodiment, the Nrp-1 antibody bindsto the a1a2 (A) domain of the Nrp-1 polypeptide.

As used herein, the term “Sema3A antibody” refers to an antibody thatspecifically binds to (interacts with) a Sema3A protein and displays nosubstantial binding to other naturally occurring proteins other than theones sharing the same antigenic determinants as the Sema3A protein.Similarly, the term “Nrp-1 antibody” refers to an antibody thatspecifically binds to (interacts with) a Nrp-1 protein and displays nosubstantial binding to other naturally occurring proteins other than theones sharing the same antigenic determinants as the Nrp-1 protein.Sema3A/Nrp-1 antibodies include polyclonal, monoclonal, humanized aswell as chimeric antibodies. The term antibody or immunoglobulin is usedin the broadest sense, and covers monoclonal antibodies (including fulllength monoclonal antibodies), polyclonal antibodies, multispecificantibodies and antibody fragments so long as they exhibit the desiredbiological activity. Antibody fragments comprise a portion of a fulllength antibody, generally an antigen binding or variable regionthereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, andFv fragments, diabodies, linear antibodies, single-chain antibodymolecules, single domain antibodies (e.g., from camelids), shark NARsingle domain antibodies, and multispecific antibodies formed fromantibody fragments. Antibody fragments can also refer to bindingmoieties comprising CDRs or antigen binding domains including, but notlimited to, VH regions (VH, VH-VH), anticalins, PepBodies™,antibody-T-cell epitope fusions (Troybodies) or Peptibodies.

Anti-human sem3A/Nrp-1 antibodies have been previously prepared⁴³ andare also commercially available from various sources including SantaCruz.

In general, techniques for preparing antibodies (including monoclonalantibodies and hybridomas) and for detecting antigens using antibodiesare well known in the art and various protocols are well known andavailable.

b. Soluble Nrp-1 Receptor or Fragment Thereof

In accordance with the present invention, soluble Nrp-1 receptor(UniprotKB/Swiss prot 014786, isoform 2) or a functional fragmentthereof may be used to reduce Sema3A induced vascular hyperpermeability.In a particular embodiment, the soluble Nrp-1 receptor functionalfragment is a fragment which binds to Sema3A but not to VEGF. Forexample the functional fragment may comprise the a1a2 domain which bindsto Sema3A but not to VEGF.

Inhibition of Sem3A Expression

Various approaches are available for decreasing Sema3A expression andthus Sema3A induced vascular hyperpermeability in the retina whichcontributes to macular edema. Non-limiting example includes the use ofsmall hairpin shRNA (RNAi), antisense, ribozymes, TAL effectorstargeting the Sema3A promoter or the like.

Expression of shRNAs in cells can be obtained by delivery of plasmids orthrough viral (e.g., lentiviral vector) or bacterial vectors. In aparticular embodiment, the shRNAs which may be used in accordance withthe present invention have the following sequences.

TABLE 1 sequences of shRNAs against Sema3A. Mature SEQ Antisense IDShRNA target Sequence NO: TRCN0000058138 Human AAATCCTTGAT  3 Sema3AATTAACCAGG TRCN0000058139 Human TTTCCCGTAAA  4 Sema3A TATCACACCGTRCN0000058142 Human TTGAAACTACT  5 Sema3A TTAAGAACGG TRCN0000058140Human AAATTAGCACA  6 Sema3A TTCTTTCAGG TRCN0000067328 Mouse AAATTGCCAAT 7 Sema3A ATACCAAGGC TRCN0000067331 Mouse AATGAGCTGCA  8 Sema3ATGAAGTCTCG TRCN0000067330 Mouse AAATTGGCACA  9 Sema3A TTCTTTCAGGTRCN0000067329 Mouse TTCATTAGGAA 10 Sema3A TACATCCTGC TRCN0000067332Mouse TTATTTATAGG 11 Sema3A AAACACTGGG

Therefore, in alternative embodiments, the invention provides antisense,shRNA molecules and ribozymes for exogenous administration to effect thedegradation and/or inhibition of the translation of mRNA of interest.Preferably, the antisense, shRNA molecules and ribozymes target humanSema3A. Examples of therapeutic antisense oligonucleotide applicationsinclude: U.S. Pat. No. 5,135,917, issued Aug. 4, 1992; U.S. Pat. No.5,098,890, issued Mar. 24, 1992; U.S. Pat. No. 5,087,617, issued Feb.11, 1992; U.S. Pat. No. 5,166,195 issued Nov. 24, 1992; U.S. Pat. No.5,004,810, issued Apr. 2, 1991; U.S. Pat. No. 5,194,428, issued Mar. 16,1993; U.S. Pat. No. 4,806,463, issued Feb. 21, 1989; U.S. Pat. No.5,286,717 issued Feb. 15, 1994; U.S. Pat. No. 5,276,019 and U.S. Pat.No. 5,264,423; BioWorld Today, Apr. 29, 1994, p. 3.

Preferably, in antisense molecules, there is a sufficient degree ofcomplementarity to the mRNA of interest to avoid non-specific binding ofthe antisense molecule to non-target sequences under conditions in whichspecific binding is desired, such as under physiological conditions inthe case of in vivo assays or therapeutic treatment or, in the case ofin vitro assays, under conditions in which the assays are conducted. Thetarget mRNA for antisense binding may include not only the informationto encode a protein, but also associated ribonucleotides, which forexample form the 5′-untranslated region, the 3′-untranslated region, the5′ cap region and intron/exon junction ribonucleotides. A method ofscreening for antisense and ribozyme nucleic acids that may be used toprovide such molecules as Shc inhibitors of the invention is disclosedin U.S. Pat. No. 5,932,435.

Antisense molecules (oligonucleotides) of the invention may includethose which contain intersugar backbone linkages such asphosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages, phosphorothioates and those with CH₂—NH—O—CH₂,CH₂—N(CH₃)—O—CH₂ (known as methylene(methylimino) or MMI backbone),CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones(where phosphodiester is O—P—O—CH₂). Oligonucleotides having morpholinobackbone structures may also be used (U.S. Pat. No. 5,034,506). Inalternative embodiments, antisense oligonucleotides may have a peptidenucleic acid (PNA, sometimes referred to as “protein nucleic acid”)backbone, in which the phosphodiester backbone of the oligonucleotidemay be replaced with a polyamide backbone wherein nucleosidic bases arebound directly or indirectly to aza nitrogen atoms or methylene groupsin the polyamide backbone (Nielsen et al., 1991, Science 254:1497 andU.S. Pat. No. 5,539,082). The phosphodiester bonds may be substitutedwith structures which are chiral and enantiomerically specific. Personsof ordinary skill in the art will be able to select other linkages foruse in practice of the invention.

Oligonucleotides may also include species which include at least onemodified nucleotide base. Thus, purines and pyrimidines other than thosenormally found in nature may be used. Similarly, modifications on thepentofuranosyl portion of the nucleotide subunits may also be effected.Examples of such modifications are 2′-O-alkyl- and2′-halogen-substituted nucleotides. Some specific examples ofmodifications at the 2′ position of sugar moieties which are useful inthe present invention are OH, SH, SCH₃, F, OCN, O(CH₂)_(n) NH₂ orO(CH₂)_(n) CH₃ where n is from 1 to about 10; C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O—,S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂,heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;substituted silyl; an RNA cleaving group; a reporter group; anintercalator; a group for improving the pharmacokinetic properties of anoligonucleotide; or a group for improving the pharmacodynamic propertiesof an oligonucleotide and other substituents having similar properties.One or more pentofuranosyl groups may be replaced by another sugar, by asugar mimic such as cyclobutyl or by another moiety which takes theplace of the sugar.

In some embodiments, the antisense oligonucleotides in accordance withthis invention may comprise from about 5 to about 100 nucleotide units.As will be appreciated, a nucleotide unit is a base-sugar combination(or a combination of analogous structures) suitably bound to an adjacentnucleotide unit through phosphodiester or other bonds forming a backbonestructure.

In a further embodiment, expression of a nucleic acid encoding apolypeptide of interest (Sema3A or Nrp-1), or a fragment thereof, may beinhibited or prevented using RNA interference (RNAi) technology, a typeof post-transcriptional gene silencing. RNAi may be used to create apseudo “knockout”, i.e. a system in which the expression of the productencoded by a gene or coding region of interest is reduced, resulting inan overall reduction of the activity of the encoded product in a system.As such, RNAi may be performed to target a nucleic acid of interest orfragment or variant thereof, to in turn reduce its expression and thelevel of activity of the product which it encodes. Such a system may beused for functional studies of the product, as well as to treatdisorders related to the activity of such a product. RNAi is describedin for example published US patent applications 20020173478 (Gewirtz;published Nov. 21, 2002) and 20020132788 (Lewis et al.; published Nov.7, 2002). Reagents and kits for performing RNAi are availablecommercially from for example Ambion Inc. (Austin, Tex., USA) and NewEngland Biolabs Inc. (Beverly, Mass., USA).

The initial agent for RNAi in some systems is a dsRNA moleculecorresponding to a target nucleic acid. The dsRNA (e.g., shRNA) is thenthought to be cleaved into short interfering RNAs (siRNAs) which are21-23 nucleotides in length (19-21 bp duplexes, each with 2 nucleotide3′ overhangs). The enzyme thought to effect this first cleavage step hasbeen referred to as “Dicer” and is categorized as a member of the RNaseIII family of dsRNA-specific ribonucleases. Alternatively, RNAi may beeffected via directly introducing into the cell, or generating withinthe cell by introducing into the cell a suitable precursor (e.g. vectorencoding precursor(s), etc.) of such an siRNA or siRNA-like molecule. AnsiRNA may then associate with other intracellular components to form anRNA-induced silencing complex (RISC). The RISC thus formed maysubsequently target a transcript of interest via base-pairinginteractions between its siRNA component and the target transcript byvirtue of homology, resulting in the cleavage of the target transcriptapproximately 12 nucleotides from the 3′ end of the siRNA. Thus thetarget mRNA is cleaved and the level of protein product it encodes isreduced.

RNAi may be effected by the introduction of suitable in vitrosynthesized siRNA (shRNAs) or siRNA-like molecules into cells. RNAi mayfor example be performed using chemically-synthesized RNA.Alternatively, suitable expression vectors may be used to transcribesuch RNA either in vitro or in vivo. In vitro transcription of sense andantisense strands (encoded by sequences present on the same vector or onseparate vectors) may be effected using for example T7 RNA polymerase,in which case the vector may comprise a suitable coding sequenceoperably-linked to a T7 promoter. The in vitro-transcribed RNA may inembodiments be processed (e.g. using E. coli RNase III) in vitro to asize conducive to RNAi. The sense and antisense transcripts are combinedto form an RNA duplex which is introduced into a target cell ofinterest. Other vectors may be used, which express small hairpin RNAs(shRNAs) which can be processed into siRNA-like molecules. Variousvector-based methods and various methods for introducing such vectorsinto cells, either in vitro or in vivo (e.g. gene therapy) are known inthe art.

Accordingly, in an embodiment expression of a nucleic acid encoding apolypeptide of interest (Sema3A or Nrp-1), or a fragment thereof, may beinhibited by introducing into or generating within a cell an siRNA orsiRNA-like molecule corresponding to a nucleic acid encoding apolypeptide of interest (e.g. myostatin), or a fragment thereof, or toan nucleic acid homologous thereto. “siRNA-like molecule” refers to anucleic acid molecule similar to an siRNA (e.g. in size and structure)and capable of eliciting siRNA activity, i.e. to effect theRNAi-mediated inhibition of expression. In various embodiments such amethod may entail the direct administration of the siRNA or siRNA-likemolecule into a cell, or use of the vector-based methods describedabove. In an embodiment, the siRNA or siRNA-like molecule is less thanabout 30 nucleotides in length. In a further embodiment, the siRNA orsiRNA-like molecule is about 21-23 nucleotides in length. In anembodiment, siRNA or siRNA-like molecule comprises a 19-21 bp duplexportion, each strand having a 2 nucleotide 3′ overhang. In embodiments,the siRNA or siRNA-like molecule is substantially identical to a nucleicacid encoding a polypeptide of interest, or a fragment or variant (or afragment of a variant) thereof. Such a variant is capable of encoding aprotein having activity similar to the polypeptide of interest.

A variety of viral vectors can be used to obtain shRNA/RNAi expressionin cells including adeno-associated viruses (AAVs), adenoviruses, andlentiviruses. With adeno-associated viruses and adenoviruses, thegenomes remain episomal. This is advantageous as insertional mutagenesisis avoided. It is disadvantageous in that the progeny of the cell willlose the virus quickly through cell division unless the cell dividesvery slowly. AAVs differ from adenoviruses in that the viral genes havebeen removed and they have diminished packing capacity. Lentivirusesintegrate into sections of transcriptionally active chromatin and arethus passed on to progeny cells. With this approach there is increasedrisk of insertional mutagenesis; however, the risk can be reduced byusing an integrase-deficient lentivirus.

Pharmaceutical Compositions

The Sema3A inhibitors of the present invention can be administered to ahuman subject by themselves or in pharmaceutical compositions where theyare mixed with suitable carriers or excipient(s) at doses to treat orprevent vascular hyperpermeabilty, non-proliferative retinopathy,retinal swelling or macular edema and associated symptoms. Mixtures ofthese compounds can also be administered to the subject as a simplemixture or in suitable formulated pharmaceutical compositions. Atherapeutically effective dose further refers to that amount of thecompound or compounds sufficient to result in the prevention ortreatment of macular edema and/or associated symptoms (spotted or blurryvision, Sema3A-associated hyperpermeability, edema, retinal swelling,and/or blood retinal barrier leakage). Techniques for formulation andadministration of the compounds of the instant application may be foundin “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton,Pa., latest edition.

Routes of Administration

Suitable routes of administration may, for example, include systemic,oral and ocular (eye drops or intraocular injections). Preferred routesof administration comprise eye drops and intraocular injections. Theformulations may also be in the form of sustained release formulations.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with endothelial orcell-specific antibody.

Composition/Formulation

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in a conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen. For injection, theagents of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer.

For ocular administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierssuitable for ocular administration well known in the art.

The compounds may be formulated for ocular administration e.g., eyedrops or ocular injections bolus injection. Formulations for injectionmay be presented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Alternatively, other delivery systems for pharmaceutical compounds maybe employed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs.

Effective Dosage

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amounts is well within thecapability of those skilled in the art.

The effective dose of the compound inhibits the cellular signalingfunction of Sema3A sufficiently to reduce or prevent vascularhyperpermeability and blood retinal barrier leakage without causingsignificant adverse effects. Certain compounds which have such activitycan be identified by in vitro assays that determine the dose-dependentinhibition of Sema3A inhibitors.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellularassays. For example, a dose can be formulated in cellular and animalmodels to achieve a circulating concentration range that includes the1050 as determined in cellular assays (i e., the concentration of thetest compound which achieves a half-maximal inhibition of the cellularsignaling function of Sema3A, usually in response to inflammatorymediators such as II-1β or other activating stimulus such as hypoxia,ischemia, cellular stress, ER stress.

A therapeutically effective amount refers to that amount of the compoundthat results in amelioration of symptoms in a subject. Similarly, aprophylactically effective amount refers to the amount necessary toprevent or delay symptoms in a patient (e.g., Sema3A-induced vascularhyperpermeability, spotted and/or blurry vision, pericytes loss, macularedema, retinal swelling, blood retinal barrier leakage, etc.). Toxicityand therapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., determining the maximum tolerated dose (MTD) and the ED (effectivedose for 50% maximal response). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio between MTD and ED50. Compounds which exhibit high therapeuticindices are preferred. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition.

Dosage amount and interval may be adjusted individually to providelevels of the active compound which are sufficient to maintain theSema3A modulating effects, or minimal effective concentration (MEC). TheMEC will vary for each compound but can be estimated from in vitro data;e. g. the concentration necessary to achieve substantial inhibition ofSema3A expression or activity (e.g., binding to Nrp-1 receptor) Dosagesnecessary to achieve the MEC will depend on individual characteristicsand route of administration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

Packaging

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack or dispenser device may be accompanied byinstructions for administration. Compositions comprising a compound ofthe invention formulated in a compatible pharmaceutical carrier may alsobe prepared, placed in an appropriate container, and labeled fortreatment of an indicated condition. Suitable conditions indicated onthe label may include the prevention and treatment of macular edema suchas diabetic macular edema and age-related macular edema, retinalvascular hyperpermeability, blood retinal barrier leakage or the like.

Screening Assays

Having demonstrated that increased Sema3A activity is associated withthe BRB leakage and retinal vascular hyperpermeability, the inventionrelates to the use of Sema3A as a target in screening assays used toidentify compounds that are useful for the prevention or treatmentretinal vascular hyperpermeability (e.g., non-proliferative diabeticretinopathy, macular edema, retinal swelling, etc.), said methodcomprising determining whether:

-   -   (a) the level of expression of a Sema3A nucleic acid or encoded        polypeptide;    -   (b) the level of Sema3A activity;    -   (c) the level of a molecule generated by a Sema3A activity; or    -   (d) any combination of (a) to (c);

is decreased in the presence of a test compound relative to in theabsence of said test compound; wherein said decrease is indicative thatsaid test compound is potentially useful for the prevention andtreatment of retinal vascular hyperpermeability. In an embodiment, theabove-mentioned method is an in vitro method. In an embodiment, theSema3A activity is its binding to the Nrp-1 receptor. In a furtherembodiment, the Sema3A activity is the increased vascular permeability.

In another embodiment of the invention, a reporter assay-based method ofselecting agents which modulate Sema3A expression is provided. Themethod includes providing a cell comprising a nucleic acid sequencecomprising a Sema3A transcriptional regulatory sequence operably-linkedto a suitable reporter gene. The cell is then exposed to the agentsuspected of affecting Sema3A expression (e.g., a test/candidatecompound) and the transcription efficiency is measured by the activityof the reporter gene. The activity can then be compared to the activityof the reporter gene in cells unexposed to the agent in question.Suitable reporter genes include but are not limited tobeta(β)-D-galactosidase, luciferase, chloramphenicol acetyltransferaseand green fluorescent protein (GFP).

Accordingly, the present invention further provides a method ofidentifying or characterizing a compound for treating or preventingretinal vascular hyperpermeability, the method comprising: (a)contacting a test compound with a cell comprising a first nucleic acidcomprising a transcriptionally regulatory element normally associatedwith a Sema3A gene (e.g., a promoter region naturally associated with aSema3A gene), operably linked to a second nucleic acid comprising areporter gene capable of encoding a reporter protein; and (b)determining whether reporter gene expression or reporter proteinactivity is decreased in the presence of said test compound, saiddecrease in reporter gene expression or reporter protein activity beingan indication that said test compound may be used for treating orpreventing retinal vascular hyperpermeability (such as retinal swellingin non-proliferative diabetic retinopathy or macular edema). In anembodiment, the above-mentioned method is an in vitro method.

The above-noted assays may be applied to a single test compound or to aplurality or “library” of such compounds (e.g., a combinatoriallibrary). Any such compound may be utilized as lead compound and furthermodified to improve its therapeutic, prophylactic and/or pharmacologicalproperties for the prevention and treatment of obesity and/orobesity-related hypertension.

Such assay systems may comprise a variety of means to enable andoptimize useful assay conditions. Such means may include but are notlimited to: suitable buffer solutions, for example, for the control ofpH and ionic strength and to provide any necessary components foroptimal Sema3A activity and stability (e.g., protease inhibitors),temperature control means for optimal Sema3A activity and or stability,and detection means to enable the detection of the Sema3A and Nrp-1interaction. A variety of such detection means may be used, includingbut not limited to one or a combination of the following: radiolabelling(e.g., ³²P, ¹⁴C, ³H), antibody-based detection, fluorescence,chemiluminescence, spectroscopic methods (e.g., generation of a productwith altered spectroscopic properties), various reporter enzymes orproteins (e.g., horseradish peroxidase, green fluorescent protein),specific binding reagents (e.g., biotin/streptavidin), and others.

The assay may be carried out in vitro utilizing a source of Sema3A whichmay comprise naturally isolated or recombinantly produced Sema3A, inpreparations ranging from crude to pure. Recombinant Sema3A may beproduced in a number of prokaryotic or eukaryotic expression systems,which are well known in the art (see for example Martin F. et al., 2001.Immunogenetics 53(4): 296-306) for the recombinant expression of Sema3A.Such assays may be performed in an array format. In certain embodiments,one or a plurality of the assay steps are automated.

A homolog, variant and/or fragment of Sema3A which retains activity(e.g., it binds to the Nrp-1 receptor) may also be used in the screeningmethods of the invention. Homologues include protein sequences, whichare substantially identical to the amino acid sequence of full lengthSema3A (e.g., FIG. 1), or matured fragment, sharing significantstructural and functional homology with Sema3A. Variants include, butare not limited to, proteins or peptides, which differ from a Sema3A byany modifications, and/or amino acid substitutions, deletions oradditions (e.g., fusion with another polypeptide). Modifications canoccur anywhere including the polypeptide backbone, (i.e., the amino acidsequence), the amino acid side chains and the amino or carboxy termini.Such substitutions, deletions or additions may involve one or more aminoacids. Fragments include a fragment or a portion of a Sema3A or afragment or a portion of a homologue or variant of a Sema3A whichretains Sema3A activity, i.e., binds to the Nrp-1 receptor and causesvascular hyperpermeabilisation.

Example 1 Material and Methods

Human Samples

Approval of human clinical protocol and informed consent form byMaisonneuve-Rosemont Hospital (HMR) ethics committee and recruitment ofpatients for local core vitreal biopsy sampling from patients afflictedwith T1 DM.

Animals

All studies were performed according to the Association for Research inVision and Ophthalmology (ARVO) Statement for the Use of Animals inOphthalmic and Vision Research and were approved by the Animal CareCommittee of the University of Montreal in agreement with the guidelinesestablished by the Canadian Council on Animal Care. C57Bl/6 wild-typewere purchased from The Jackson Laboratory. Tamoxifen-inducible(Tam-inducible) Cre mice (Ter^(Cre-Esr)1; no. 004682) and Neuropilin 1floxed mice (Nrp1^(tm2Ddg)/J; no. 005247) were purchased from TheJackson Laboratory.

Streptozotocin (STZ) Mouse Model

C57BL/6J mice of 6- to 7-week were weighted and their baseline glycemiawas measured (Accu-Chek, Roche). Mice were injected intraperitoneallywith streptozotocin (Sigma-Alderich, St. Louis, Mo.) for 5 consecutivedays at 55 mg/Kg. Age-matched controls were injected with buffer only.Glycemia was measured again a week after the last STZ injection and micewere considered diabetic if their non-fasted glycemia was higher than 17mM (300 mg/dL).

Real-Time PCR Analysis

RNA was isolated using the GenElute™ Mammalian Total RNA Miniprep Kit(Sigma) and digested with DNase I to prevent amplification of genomicDNA. Reversed transcription was performed using M-MLV reversetranscriptase and gene expression analyzed using SybrGreen™ in an ABIBiosystems™ Real-Time PCR machine. β-actin was used as a reference gene.

Laser-Capture Microdissection

Eyes were enucleated from P14 pups in OIR (oxygen induced retinopathy)or normoxic littermates and flash-frozen in OCT. We then cut 12 μmsections using a Leica cryostat at −20° C. and air-dried for 10 min. Wedissected retinal layers using a Zeiss Observer microscope equipped witha Palm MicroBeam™ device for laser-capture microdissection. We isolatedmRNA from these sections and performed qPCRs as described above.

Western-Blotting

For assessment of retinal protein levels, we enucleated eyes at varyingtime points and rapidly dissected and homogeneized retinas. Proteinconcentrations were assessed by BCA assay (Sigma), and then 30 ug ofprotein analyzed for each condition by standard SDS-PAGE technique.Antibodies used for Western-blotting are: Nrp-1 (R&D Systems, #AF566),pVE-Cadherin (Invitrogen, #441145G), Src (Cell Signaling, #2108), pSRC(Cell Signaling, #2101), FAK (Cell Signaling, #3285), pFAK (CellSignaling, #3281), b-Actin (Sigma, #A2228), Sema3A (Santa Cruz, #sc-1148OR ABCAM #ab23393).

Immunohistochemistry

To localize protein expression, eyes were enucleated from mice and fixedin 4% paraformaldehyde at room temperature for 4 h at RT and incubatedin 30% sucrose overnight and then frozen in OCT compound. We thenembedded the whole eye in optimal cutting temperature compound at −20°C. and performed 12 um serial sections. We carried outimmunohistochemistry experiments and visualized the sections with anepifluorescent microscope (Zeiss AxioImager™) or confocal microscope(Olympus confocal FV1000). Antibodies used for immunohistochemistry are:Sema3A (ABCAM #ab23393), Smooth Muscle Actin (SMA) (ABMCA, #ab7817) andβIII-tubulin (ECM). Secondary antibodies are Alexa 594 (Invitrogen,#A11005) and Alexa 488 (Invitrogen, #A11008).

For visualization of pan-retinal vasculature, flatmount retinas werestained with stained with fluoresceinated Isolectin B4 (Alexa Fluor594-I21413, Molecular Probes) in 1 mM CaCl₂ in PBS for retinalvasculature. For assessment of vascular permeability (see EvansBlue—EB—permeation), we injected mice vitreally with Vehicle and VEGF,after 2 hours of EB injection, the eyes were harvested and retinas weredissected for flatmount or prepared for cryosections and visualizationunder a fluorescent microscope

Preparation of Lentivirus

We produced infectious lentiviral vectors by transfecting lentivectorand packaging vectors into HEK293T cells (Invitrogen) as previouslydescribed⁴⁰. Viral supernatants were concentrated byultra-centrifugation (>500-fold) and titers determined by ELISA forviral p24 antigen using a commercial kit (Clonetech).

Soluble Recombinant NRP1 and Mouse Anti-VEGF

STZ treated diabetic C57BL/6J mice were intravitreally injected withrmNRP1 from plasmid (Mamluk et al., 2002¹⁷) or R&D Systems at 6 and 7weeks after STZ administration. Specific mouse anti-VEGF was purchasedfrom R&D Systems (AF-493-NA) and 1 μl was injected at 80 ug/mL. RetinalEvans blue permeation assay was performed at 8 weeks after STZ treatmentas described above.

Statistical Analyses

Data are presented as mean±s.e.m. We used Student's T-test and ANOVA,where appropriate, to compare the different groups; a P<0.05 wasconsidered statistically different.

Example 2 Sema3A is Elevated in the Vitreous of Human Patients Sufferingfrom Diabetic Retinopathy

In order to evaluate the potential role of Sema3A in mediating theedematous phenotype observed in DR, we first sought to determine thepresence of this guidance cue in the vitreous of patients suffering fromDME. Vitreous was recovered during standard vitroretinal surgery from 8patients. Five samples were obtained from T1 DM patients suffering fromDME and 3 from control patients (non-vascular pathology) undergoingsurgery for macular hole (MH) or Epiretinal Membrane (ERM).

Western blot analysis revealed that both pro-(˜125 kDa) and active (˜95kDa) forms of Sema3A were robustly induced in patients affected by DME(FIG. 1a,b ). Consistent with a prospective role in DME,ELISA-baseddetection of Sema3A revealed a significant increase in the vitreous ofpatients suffering from DME when compared to nonvascular ocularpathologies (control median 3.79 ng/ml [interquartile range {IQR}: 25%,75%: 2.08 ng/ml, 5.58 ng/ml]; DME median 16.27 ng/ml [IQR: 25%, 75%:5.770 ng/ml, 35.36 ng/ml]; p=0.0464) (Data not shown). Spectral-domain.Optical Coherence Tomography (OCT) was performed and three-dimensional(3D) maps were generated to evaluate the extent of retinal damage andedema. In contrast to controls sampled DME patients showed significantretinal swelling, specifically in the macular and peri-macular zones asshown in FIG. 1c,d . Detailed DME patient characteristics are presentedin FIG. 1 e.

These data provide the rational to explore the role of Sema3A in thecontext of diabetes-induced retinal vasculopathy.

Example 3 Neuronal Sema3A is Upregulated in the Early Phases ofStreptozotocin-Induced Diabetes

Given the elevated levels of Sema3A in the vitreous of DME patients,Applicant sought to elucidate the dynamics and pattern of Sema3Aexpression in a mouse model of type 1 diabetes mellitus (T1DM).Streptozotocin (STZ) was administered over 5 consecutive days to6-week-old C57BL/6J mice, and glycemia was monitored according to thescheme depicted in FIG. 2a . Mice were considered diabetic if theirnon-fasted glycemia was higher than 17 mM (300 mg/dL).

As early as 4 weeks after induction of diabetes, retinal levels ofSema3A where over 2-fold higher in STZ treated mice when compared tovehicle injected controls (p=0.0045, n=5). These significantly higherretinal levels of Sema3A persisted at 8 weeks (Sema3A, 2.80±0.340;p=0.0011; VEGF, 1.236±0.193; p=0.266, n=8), 12 weeks (Sema3A,4.07±0.798; p=0.00846; VEGF, 0.923±0.145; p=0.612, n=4), and 14 weeks(Sema3A, 2.44±0.593; p=0.0334; VEGF, 3.26±0.65; p=0.0253, n=3).Importantly, throughout early time points of disease (4-12 weeks), VEGFlevels in STZ-treated mice remained at similar levels to that observedin vehicle treated congener mice as has been previously described ²⁴(FIG. 2b ). At all analyzed time-points, STZ-treated mice showedpathologically elevated blood glucose levels of ˜30 mM (FIG. 2d ;p<0.0001 for both 8 and 4 weeks of diabetes). Importantly, the rise inexpression of Sema3A was an early event and preceded pericyte loss asboth STZ and vehicle-treated mice showed similar levels of smooth muscleactin (SMA, FIG. 2c ). Similarly, expression levels of the tightjunction components occludin and claudin-5 varied minimally at the earlytime of 8 weeks (FIG. 2e ). Finally mice both STZ- and vehicle-treatedmice showed no significant difference in transcript levels for pericytemarkers platelet-derived growth factor receptor-b (Pdgfr-b; 1.477±0.364;p=0.219, n=11), NG2 proteoglycan (Ng2; 2.065±0.886; p=0.316, n=4), oralpha smooth muscle actin (a-Sma; 1.342±0.441; p=0.494, n=4).

Example 4 The Expression Pattern of Sema3A is Geographically Consistentwith a Role in Diabetic Retinopathy

Applicant next sought to determine the cellular source of Sema3A in thediabetic retina. Immunohistochemistry on retinal cryosections revealedthat Sema3A was strongly expressed by retinal neurons of the ganglioncell layer (GCL and inner-nuclear layer (INL) (FIG. 2f,g ). The mostprominent expression was noted in retinal ganglion cells (RGCs) asdemonstrated by co-localization with the RGC marker pIII-tubulin.Consistent with the retinal immuno-localization of Sema3A, laser-capturemicro-dissection of retinal layers from normal and diabetic micefollowed by quantitative RT-PCR pinpointed Sema3A to neurons in closeproximity to vascular beds (FIG. 2h ).

Example 5 Retinal Barrier Function is Compromised by Sema3A

Given the observed rise in retinal Sema3A levels in diabetes, Applicantproceeded to investigate the propensity of Sema3A to disrupt vascularbarrier function. Intravitreal injection of Sema3A resulted in a ˜2-foldincrease (FIG. 3a ; p<0.01) in retinal vascular permeability asdetermined by Evans Blue (EB) permeation. This increase was similar tothat observed with intravtireal administration of VEGF (FIG. 3a ;p<0.05) or a combination of both Sema3A and VEGF (FIG. 3a ; p<0.01).FIG. 3b depicts confocal images of retinal sections injected withvehicle, VEGF and Sema3A, showing the representative increased patternof EB leakage. To further examine the ability of Sema3A to compromiseendothelial barrier function, Applicant carried out real-time analysisof trans-endothelial electric resistance. Treatment of an intactendothelial monolayer with Sema3A reduced barrier function in amagnitude similar, yet lower than VEGF in the first 6 hours followingaddition (FIG. 3c ).

Applicant next ascertained that Sema3A activated signaling pathwaysknown to promote vascular permeability. In this respect Applicantinvestigated, by Western blot analysis, the activation profiles of Srcand focal adhesion kinase (FAK) known to transduce extracellular signalsthat provoke the loosening of endothelial cell tight junctions²⁵⁻²⁸.Stimulation of Human Retinal Microvascular Endothelial Cells (HRMECs) byeither Sema3A or VEGF lead to robust phosphorylation of Src at Tyr416 inthe activation loop of the kinase domain which is reported to enhanceenzyme activity²⁹. In turn, FAK was phosphorylated on Tyr576 and 577(sites for Src-kinases). Ultimately, the tight junction proteinsVE-cadherin became phosphorylated respectively on tyrosine-731 (siteassociated with increased vascular permeability³⁰⁻³²) (FIG. 3d ).Consistent with the above data on retinal permeability (FIG. 3a ), anadditive or enhanced effect was not observed when simulation wasperformed with a combination of Sema3A and VEGF suggesting that bothfactors signal via redundant pathways (FIG. 3d ). In accordance toVE-cadherin western blot analysis (FIG. 3d ), flatmounted retinasinjected with Sema3A or VEGF showed higher VE-cadherin phosphorylationat Y731 (arrows) than vehicle-injected retinas in co-localization withlectin stained retinal vessels (FIG. 3g ). Similarly, retinal flatmountsfrom STZ-injected and vehicle-injected mice showed VE-cadherinphosphorylation colocalizing with retinal vessels (FIG. 3h ).

Consistent with a role in disrupting barrier function, confocalmicroscopy of Sema3A-treated HRMECs revealed pronounced formation ofvascular retraction fibers as determined by VE-cadherin and phalloidinstaining (white arrows; FIG. 3f ). The observed retraction was similarto that with VEGF alone or with a combination of VEGF and Sema3A.Importantly, at the doses employed in the instant study (100-200 uM)Sema3A did not induce cell death or apoptosis as determined byassessment of activation of caspase-3 (FIG. 3i ). These data underscorethe direct effect on retinal vascular permeability of Sema3A.

Example 6 Inhibition of Neuron-Derived Sema3A Efficiently ReducesVascular Permeability in T1 DM

Recent studies demonstrate that retinal neurons may exert an importantinfluence on the blood vessels that perfuse them ^(14,33-35). In lightof the robust expression of Sema3A in RGCs and the INL as well as itsability to promote vascular leakage, Applicant sought to inhibitproduction of this guidance cue directly in these cell populations. Tospecifically block Sema3A production in RGCs or neurons of the INL invivo, lentiviral (Lv) vectors carrying a shRNA against Sema3A weregenerated (TTATTTATAGGAAACACTGGG-SEQ ID NO:11). These Lv vectors with aVSVG capsid exhibit high tropism for RGCs and cells of the ONL whendelivered intravitreallyl^(14,35) (FIG. 4a ). While STZ-treated miceshow a 56.8% increase in permeability (FIG. 4b ; p<0.05, n=4) a singleintravitreal injection of Lv.shSema3A at 5 weeks of diabetes lead to asignificant 62.3% reduction in retinal Sema3A expression (FIG. 4c ;p<0.005, n=3) and provoked a proportional 49.5% decrease in vascularleakage (FIG. 4d ; p<0.05, n=3). Hence, directly targeting Sema3Aexpression in neurons of the GCL and the INL where Sema3A was mostabundantly expressed in diabetic mice (FIG. 2f-h ) effectively reducedpathological vascular leakage.

Example 7 Intravitreal Neutralization of Sema3A Reduces Retinal VascularPermeability

In order to neutralize vitreal Sema3A, we employed recombinant(r)soluble Nrp-1 as a bivalent trap for both Sema3A and VEGF. Neuropilin-1is a single-pass receptor with its extracellular domain subdivided intodistinct sub-domains of which a1a2 binds semaphorin and b1b2 bindsVEGF³⁶ (FIG. 4e ). Intravitreal injection of rNrp-1 in STZ mice at week6 and 7 after induction of diabetes lead to a 48.1% reduction in retinalpermeability measured at week 8 of diabetes (FIG. 4f ; p<0.05, n=5); asimilar magnitude to that observed with gene silencing of Sema3A (FIG.4d ). Importantly, neutralization of VEGF with a neutralizing antibodyfor mouse VEGF₁₆₄ was not effective at reducing vascular permeability atthis early stage of diabetes (vehicle vs anti-VEGF: 0.975±0.0707;P=0.7302 II rmNRP1 vs anti-mVEGF: P=0.035, n=5 distinct experiments witha total of 14 mice). This is likely attributed to the fact that VEGF isnot increased in diabetic retinas at this early time point (8 weeks)while Sema3A is robustly induced (FIG. 2b ). Together, these dataindicate that neutralization of SEMA3A in the diabetic retina is aneffective strategy to reduce vasogenic edema.

Example 8 Conditional Knockout of Nrp-1, Prevents Sema3A-Induced RetinalBarrier Function Breakdown

In light of Nrp-1 being the receptor for Sema3A, Applicant sought todetermine whether knockout of Nrp-1 protects against Sema3A-inducedvascular permeability. Because systemic germline deletion of Nrp-1 isembryonic lethal ³⁷⁻³⁹, a whole-animal tamoxifen-inducible(Tam-inducible) Cre mouse (Tg^(Cre-Esr1)) was generated to induce Nrp-1exon 2 deletion. To validate Cre recombination at the Nrp-1 locus andconfirm disruption of Nrp-1 in vivo, Tg^(Cre-Esr1); Nrp1^(fl/fl) mice(iKO) and littermates were administered Tam or vehicle (Veh) at 6 weeksof age. Systemic administration of Tam over a period of 5 consecutivedays lead to an efficient knockout of Nrp-1 in the vascular system asdetermined by Western blot (FIG. 5a ) and qPCR (FIG. 5b : P=0.0012) andresulted in near complete absence of NRP1 in retinal vessels (assessedby immunofluorescence of retinal cryosections-data not shown).Importantly, Tam-treated iKO (Tam iKO) mice showed no difference in bodyweight, size or open-field activity compared with littermates from 4through 20 weeks of age (data not shown). As expected, Tam treatedTgCre-Esr1;Nrp1fl/fl mice with disrupted Nrp-1 were protected againstSema3a-induced vascular permeability following intravitreal injection ofSema3A (1.276±0.2901; P=0.36; n=7 distinct experiments with 21 mice)(FIG. 5c ; while control Tam-treated TgCre-ESR1/Nrp1+/+ mice showed3-fold higher vascular leakage in response to Sema3A (2.972±0.2045;P=0.00065; n=3 distinct experiments with a total of 9 mice). Conversely,disruption of Nrp1 did not influence VEGF-induced vascular retinalpermeability (Tam-treated TgCre-Esr1/Nrp1fl/fl—Vehicle vs VEGF:1.814±0.1188, P=0.0024, n=3 distinct experiments with a total of 9 mice;Tam-treated TgCre-Esr1/Nrp1+/+—Vehicle vs VEGF: 1.783+0.2440; P=0.032,n=3 distinct experiments with a total of 9 mice) (FIG. 5d ) indicatingthat VEGF-induced retinal vascular permeability does not require NRP1.This is in accordance with previous work. Efficiency of sh-mediatedknockdown of Nrp1 was validated by qPCR (data not shown). Collectively,these data confirm that Sema3A-mediated inner-blood retinal barrierfunction breakdown is NRP1-dependent and validate NPR-1 as a good targetfor reducing Sema3A-mediated hyperpermeability and blood brain barrierleakage in macular edema.

Although various embodiments of the invention are disclosed herein, manyadaptations and modifications may be made within the scope of theinvention in accordance with the common general knowledge of thoseskilled in this art. Such modifications include the substitution ofknown equivalents for any aspect of the invention in order to achievethe same result in substantially the same way. In the claims, the word“comprising” is used as an open-ended term, substantially equivalent tothe phrase “including, but not limited to”. The following examples areillustrative of various aspects of the invention, and do not limit thebroad aspects of the invention as disclosed herein.

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The invention claimed is:
 1. A method of preventing or treating macularedema in a subject with non-proliferative retinopathy comprisinginhibiting Semaphorin 3A (Sema3A)-mediated ocular vascularhyperpermeability by administering a prophylactically or therapeuticallyeffective amount of at least one Sema3A antagonist selected from thegroup consisting of: i) an anti-Sema3A antibody; ii) ananti-Neuropilin-1 (Nrp-1) antibody; iii) A Sema3A antisense or shRNA;iv) a Nrp-1 antisense or shRNA; and v) a soluble Nrp-1 polypeptide orfragment thereof.
 2. The method of claim 1, wherein said subject suffersfrom a) Type II diabetes mellitus (T2DM); b) early stages of diabetesprior to Vascular Endothelial Growth Factor (VEGF), inducement; and/orc) retinal barrier swelling.
 3. The method of claim 1, wherein saidsubject has normal levels of VEGF.
 4. The method of claim 1, whereinsaid subject does not suffer from pericytes loss.
 5. The method claim 1,wherein said subject is asymptomatic.
 6. The method of claim 1, whereinsaid a) anti-Nrp-1 antibody or soluble Nrp-1-polypeptide or fragmentthereof does not reduce VEGF binding to Nrp-1; b) anti-Nrp-1 antibodybinds to the a1a2 domain of Nrp-1; c) Nrp-1 polypeptide fragmentconsists of the a1a2 domain of Nrp-1; d) anti-Sema3A antibody binds tothe Nrp1 binding domain of Sema3A; or e) anti-Sema3A shRNA comprises asequence as set forth in SEQ ID NO: 3, 4, 5 or
 6. 7. The method of claim1, wherein said Sema3A antagonist specifically targets neurons in theganglion cell layer (GCL) or inner nuclear layer (INL).
 8. The method ofclaim 6, wherein said Sema3A antagonist specifically targets neurons inthe ganglion cell layer (GCL) or inner nuclear layer (INL).
 9. Themethod of claim 1, wherein said Sema3A antagonist is a Sema3A shRNA in alentiviral vector.
 10. The method of claim 1, wherein said Sema3Aantagonist is administered intravitreally.
 11. The method of claim 1,wherein said Sema3A antagonist is an Nrp-1 polypeptide or fragmentthereof and wherein said Nrp-1 polypeptide or fragment thereof isadministered intravitreally.
 12. The method of claim 1, wherein saidSema3A antagonist is an Nrp-1 polypeptide or fragment thereof andwherein said Nrp-1 polypeptide or fragment thereof is administeredintravitreally prior to VEGF-inducement.
 13. The method of claim 6,wherein said Sema3A antagonist is an Nrp-1 polypeptide or fragmentthereof lacking Nrp-1 domain b1, b2 or b1 and b2 of Npr-1 and whereinsaid Nrp-1 polypeptide or fragment thereof is administeredintravitreally.